The present invention relates to a nucleic acid molecule which codes for the cephalosporin acetylesterase from Bacillus subtilis ATCC 6633 (DSM 11909), vectors and host cells which comprise such a nucleic acid molecule, a process for the recombinant preparation of cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11909) using said nucleic acid molecule, and a process for preparing 3-deacetylcephalosporin compounds.
Cephalosporin C is cleaved by cephalosporin acetylesterase (CAE) to 3-deacetyl-cephalosporin C (see, for example, B. J. Abbott et al., Appl. Microbiol. (1975), 413-419; J. Konecny et al., Biochim. Biophys. Acta 485 (1977), 367-378). It is furthermore possible to convert the cephalosporin compound 7-aminocephalosporanic acid (7-ACA) with CAE to 7-amino-3-deacetylcephalosporanic acid (HACA). 3-Deacetylcephalosporin C and HACA are used as intermediates in preparation of semisynthetic cephalosporins (S. Tsushima et al., Chem. Pharmacol. Bull. 27 (1979), 696-702). Various cephalosporin acetylesterases from diverse Bacillus subtilis (B. subtilis hereinafter) strains have been disclosed in the literature (see T. B. Higherd et al., J. Bacteriol. 114 (1973), 1184-1192; B. J. Abbott et al., Appl. Microbiol. (1975), 413-419; A. Takimoto et al., J. Ferment. Bioeng. 77 (1994), 17-22). Another cephalosporin acetylesterase was isolated by J. Konecny et al. (see J. Konecny et al., Biochim. Biophys. Acta 485 (1977), 367-378) from the B. subtilis strain ATCC 6633. The last-mentioned cephalosporin acetylesterase is particularly suitable for carrying out the abovementioned conversions of cephalosporin C into 3-deacetylcephalosporin C and of 7-ACA into HACA.
It is desirable, for technical and commercial reasons, to prepare the CAE required for the said conversion by a recombinant route. In particular, it would be possible in this way to produce in a simple and cost-effective manner the amounts of CAE required for carrying out the conversion process industrially. However, it has not been possible to date to clone the nucleic acid sequence coding for the cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11909).
It is thus an object of the present invention to provide the nucleic acid sequence coding for the cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 1 1909). It is a further object of the present invention to provide vectors, in particular expression vectors, which comprise this nucleic acid sequence, and host cells which comprise these vectors. It is a further object of the present invention to provide a recombinant process for preparing the said cephalosporin acetylesterase. Finally, it is an object of the present invention to provide a process for preparing 3-deacetylcephalosporin compounds using a recombinant cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11 909).
It has surprisingly been possible within the scope of the present invention to establish the nucleic acid sequence codings for the cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11909).
One aspect of the present invention is thus a nucleic acid molecule which codes for the cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11909).
In this connection, the cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11909) essentially corresponds to the activity described by J. Konecny et al. (see above). In particular, J. Konecny et al. refer to a value of the Michaelis-Menten constant Km for the conversion of cephalosporin C into 3-deacetylcephalosporin C of about Km=15 mM (at 25xc2x0 C.).
The cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11909) is, in particular, a polypeptide which has the amino acid sequence shown in the sequence identity No. (abbreviated to SEQ ID NO. hereinafter) 1 which is reproduced in Annex 1.
The nucleic acid molecule according to the invention preferably comprises a base sequence of the SEQ ID NO. 2 depicted in Annex 2. Also preferred is a nucleic acid molecule according to the invention which has exclusively the base sequence shown in SEQ ID NO. 2. A nucleic acid molecule according to the invention is, in particular, in the form of a DNA, for example of a cDNA. However, the invention also relates to nucleic acid molecules having the said properties, for example, an RNA, for example an mRNA or a pre-mRNA.
A nucleic acid molecule according to the invention can be obtained in the following way:
(i) Chromosomal DNA is extracted from B. subtilis ATCC 6633 (DSM 11909), purified and prepared for subsequent amplification, for example with the aid of PCR experiments (PCR =polymerase chain reaction). Primers able specifically to hybridize with the 5xe2x80x2 end or with the 3xe2x80x2 end of the nucleic acid sequence depicted in SEQ ID NO. 2 are constructed. An example of a 5xe2x80x2 primer which can be used is an oligonucleotide which corresponds to nucleotides Nos 1 to 27 in the nucleic acid sequence depicted in SEQ ID NO. 2. An example of a 3xe2x80x2 primer which can be used is an oligonucleotide which is complementary to nucleotides Nos 93 i to 957 in SEQ ID NO. 2. It is possible and advantageous to provide one or more restriction cleavage sites in the primers for subsequent cloning. The primers are used to carry out a suitable amplification method, for example a PCR experiment. (ii) The result of the amplification, for example of the PCR, is analysed, for example by electrophoresis on an agarose gel. It is possible with the aid of the abovementioned primers to obtain DNA fragments which can be extracted from the agarose gel and cloned into suitable vectors, for example selective plasmid vectors. A vector obtained in this way can then be used to transform a suitable host cell or a suitable host organism. For example, the DNA fragments obtained in the preceding step are inserted into an E. coli cloning vector, and the resulting recombinant plasmid is introduced by transformation or electroporation into E. coli cells. To produce colonies, the transformants are plated out on agar medium with an antibiotic, which is appropriate for the plasmid vector used, as selection pressure. (iii) A few of the resulting clones are selected, and the base sequence of the DNA insert is established. For example, DNA of the recombinant plasmid is extracted from a large number of selected E. coli colonies and is analysed with restriction enzymes. Subsequently, as a check, the base sequence of the DNA fragment originating from B. subtilis is determined. For this purpose, the fragments from several independently isolated clones are sequenced in order to detect any mutations due to the process. The nucleic acid sequence which has been established, and the amino acid sequence derived therefrom, is then compared with the sequences depicted in SEQ ID NO. 2 and SEQ ID NO. 1, respectively, in order to find a nucleic acid sequence which is sought. The appropriate clone of the host cell can then be grown further, and the vector containing the nucleic acid molecule according to the invention and, finally, the nucleic acid molecule according to the invention itself can be isolated.
Particularly suitable host cells or host organisms are bacterial strains, for example, E. coli. It is possible and advantageous to use as E. coli host a strain of the E. coli derivative K2, for example, W3110, HB101, C600, JM87, JM103, JM105 or JM109. Examples of E. coli vectors which can be used for the cloning are both plasmid vectors such as pUC13, pK19, pBR322 and pAT153, and phage vectors, for example, lambda gt10. The abovementioned hosts and vectors are merely examples of many others which are commercially available and can be obtained straightforwardly.
Oligonucleotides can be synthesized using a commercially available DNA synthesizer in accordance with the manufacturer""s instructions.
Determination of a nucleic acid sequence according to the invention can be carried out by the method of Sanger et at. (1977), employing a known M13 vector system, or a commercially available sequencing kit can be used. Alternatively, a nucleic acid sequence according to the invention can be established using an automated sequencer.
The recombinant processes described above for preparing a nucleic acid molecule according to the invention are familiar to the skilled person. In this connection, reference is made to the monograph by J. Sambrook et al. (Ed.), xe2x80x9cMolecular Cloningxe2x80x9d (1989), Cold Spring Harbor Laboratory Press.
Alternatively, a nucleic acid molecule according to the invention can also be prepared semisynthetically by known processes. For this purpose, for example, complementary nucleic acid fragments which overlap in the 5xe2x80x2 or 3xe2x80x2 region and have in each case a length of up to 250-300 nucleotides of SEQ ID NO.2 are prepared and hybridized together, and the gaps are filled in enzymatically. Double-stranded nucleic acid fragments obtained in this way are finally, where appropriate, ligated together (see, for example, H. lbelgaufts, xe2x80x9cGentechnologie von A bis Zxe2x80x9d (1 990), VCH, Weinheim),
A further aspect of the present invention relates to a vector which is compatible with a predetermined host cell, the vector comprising a nucleic acid molecule according to the invention. As described above, a vector of this type is, in a preferred embodiment, a cloning vector.
In a further preferred embodiment, a vector according to the invention is an expression vector. An expression vector of this type can be used in a process for the recombinant preparation of the cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11909), a process of this type representing a further aspect of the invention. A process of this type according to the invention comprises,
(i) a host cell which is transformed with an expression vector according to the invention being cultivated under conditions suitable for bringing about the expression of the cephalosporin acetylesterase and, where appropriate,
(ii) the expressed cephalosporin acetylesterase being isolated.
To construct a recombinant expression vector, the nucleic acid fragment according to the invention, in particular a corresponding cDNA, is modified in a suitable manner for cloning and then introduced into a gene expression vector, for example an expression plasmid, so that the structural gene comes under the control of appropriate regulatory sequences, for example a suitable promoter.
Construction of an expression vector for efficient expression of the required gene in a host can be carried out by cloning a nucleic acid molecule according to the invention, in particular an appropriate DNA or cDNA, into an expression vector which is compatible with an appropriate host. A suitable host is, preferably, E. coli, preferably a derivative of the E. coli strain K12, for example a strain such as E. coli W3110 (ATCC 27325), JM103 (C. Yanish-Perron et al., Gene 33 (1985), 103-109), JM109 (C. Yanish-Perron et al., 1985, ibid.), JM83 (C. Yanish-Perron et al., 1985, ibid.), HB101 (J. Sambrook et al., 1989, ibid.), and C600 (J. Sambrook et al., 1989, ibid.). Examples of suitable expression vectors are vectors, in particular plasmids, for example pKK223-2 (GenBank Acc. No. M77749), pK19 (R. D. Pridmore, Gene 56 (1987), 309-312), or pPLa832 (E. L. Winnacker, Gene and Klone, Verlag Chemie, Weinheim 1984/1985), which comprise a suitable promoter functioning in a host (for example Lac, Tac, Trc, Trp or PL) and a ribosome binding site (RBS, Shine-Dalgamo (SD) sequence), or ATG vectors (for example pKK233-2, obtainable from
Pharmacia), which additionally comprise the translation start codon ATG. Introduction of the expression vector into a suitable host results in a microorganism which effectively expresses cephalosporin acetylesterase.
The recombinant expression vector obtained in this way is introduced by transformation into an appropriate host, producing a novel host strain producing cephalosporin acetylesterase.
The transformed cells are cultivated under suitable conditions under which expression of CAE is brought about. After expression of the CAE has taken place, the host cells are disrupted. The expressed cephalosporin acetylesterase can be purified by a conventional purification process, for example by centrifugation, column chromatography and the like or a suitable combination thereof. An alternative possibility is also, for example, to employ cell supernatents or suspensions of disrupted host cells which contain the expressed CAE subsequently.
The expressed recombinant CAE can also be purified by affinity chromatography using immobilized antibodies which themselves can be obtained in a manner known per se using CAE isolated from B. subtilis ATCC 6633 (DSM 1 1909) as antigen.
The recombinantly prepared CAE can be immobilized on solid supports, which is a great advantage especially for industrial use. The use of solid catalysts produced in this way presents great economic advantages in industrial use.
The recombinant CAE is used according to the invention in a process for preparing 3-deacetylcephalosporin compounds and subsequently for preparing semisynthetic cephalosporin compounds, in which case the acetyl radical of the acetoxymethyl group of cephalosporin compounds which have an acetoxymethyl group in position 3 is cleaved off by the CAE.
A process according to the invention of this type comprises the following steps:
(i) Conversion of a cephalosporin compound which has an acetoxymethyl group in position 3 using a recombinantly prepared cephalosporin acetylesterase from B. subtilis ATCC 6633 (DSM 11909), which is employed in free form or in immobilized form; and, where appropriate,
(ii) isolation of the 3-deacetylcephalosporin compound formed.
In order subsequently to prepare further cephalosporin compounds as final products, the 3-deacetylcephalosporin compound which has been formed is isolated, further purified where appropriate, and subsequently converted into the required final product. Alternatively, the resulting reaction solution containing 3-deacetylcephalosporin compound formed is subsequently converted in order finally to obtain the required final product.
The cephalosporin compound which is employed in the process according to the invention and has acetoxymethyl group in position 3 is, in particular, 7-aminocephalosporanic acid or a 7-acyl derivative. One example of such a derivative is cephalosporin C.
Thus, according to the invention, for example 7-aminocephalosporanic acid is converted into 7-amino-3-deacetylcephalosporanic acid and cephalosporin C is converted into 3-deacetylcephalosporin C.
The reaction conditions are familiar to the skilled person and correspond to those for the conversion with native cephalosporin acetylesterase (see, e.g., J. Konecny, Enzyme Engineering 4 (1978), pp. 253-259).
The full contents of the texts mentioned are incorporated herein by reference.